US20230313239A1 - Novel cis-aconitate synthesis enzyme and uses thereof - Google Patents

Novel cis-aconitate synthesis enzyme and uses thereof Download PDF

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US20230313239A1
US20230313239A1 US18/192,868 US202318192868A US2023313239A1 US 20230313239 A1 US20230313239 A1 US 20230313239A1 US 202318192868 A US202318192868 A US 202318192868A US 2023313239 A1 US2023313239 A1 US 2023313239A1
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itaconate
seq
prpd
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aconitate
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Gyoo Yeol Jung
Jae Seong YANG
Myung Hyun NOH
Dae Yeol YE
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Postech Research and Business Development Foundation
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Definitions

  • the present disclosure relates to a novel cis-aconitate synthesis enzyme, and more particularly, to a recombinant microorganism for producing itaconate including a cis-aconitate synthesis enzyme variant.
  • Itaconate (itaconic acid), dicarboxylic acid composed of 5 carbons, is a material that may be used as precursors of various polymer materials such as latex and plastics, drug transport, and water purification based on structural characteristics of the material. Due to great industrial potential, the itaconate was selected as one of 12 bio-based platform chemicals selected by the United States Department of Energy in 2004.
  • the present inventors have developed a novel cis-aconitate synthesis enzyme through an evolutionary design rather than existing enzymes with respect to the synthesis of cis-aconitate, an intermediate in the TCA cycle to intend to increase the efficiency of itaconate synthesis by separating existing competing TCA cycle and itaconate synthesis according to the activity of the novel enzyme.
  • the present inventors confirmed that a recombinant microorganism introduced with the enzyme responsible for the biosynthetic reaction had excellent itaconate production capacity and then completed the present disclosure.
  • the present disclosure has been made in an effort to provide an expression cassette for introducing an itaconate production pathway including a 2-methylcitrate dehydratase (PrpD) gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17.
  • PrpD 2-methylcitrate dehydratase
  • the present disclosure has also been made in an effort to provide a recombinant vector for producing an itaconate production pathway including the expression cassette.
  • the present disclosure has also been made in an effort to provide a recombinant microorganism for producing itaconate into which a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • the present disclosure has also been made in an effort to provide a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • An embodiment of the present disclosure provides an expression cassette for introducing an itaconate production pathway including a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17.
  • Another embodiment of the present disclosure provides a recombinant vector for producing itaconate including the expression cassette for introducing the itaconate production pathway.
  • Yet another embodiment of the present disclosure provides a recombinant microorganism for producing itaconate into which a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • Still another embodiment of the present disclosure provides a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • novel aconitate synthesis enzyme of the present disclosure and the recombinant microorganism introduced with the aconitate synthesis enzyme can increase the economic feasibility of itaconate, and thus can be used in various industrial fields, such as synthetic resins, latexes, and food additives in which itaconate is used.
  • FIG. 1 is a diagram illustrating an itaconate production pathway through introduction of a novel cis-aconitate synthesis enzyme in E. coli according to the present disclosure and a carbon flow compartmentalization according to the activity of the enzyme (Left: existing itaconate production pathway, Right: novel itaconate production pathway according to the present disclosure).
  • FIG. 2 is a diagram illustrating a comparison of a reaction mechanism and a reaction substrate of 2-methylstarate dehydratase (PrpD) and aconitase, which are wild types of the novel cis-aconitate synthesis enzyme according to the present disclosure.
  • PrpD 2-methylstarate dehydratase
  • aconitase which are wild types of the novel cis-aconitate synthesis enzyme according to the present disclosure.
  • FIG. 3 A is a diagram illustrating results of analyzing citrate and itaconate production of a WAICP strain according to the present disclosure ( ⁇ : Acetate (g/L), ⁇ : Cell biomass (g DCW/L), ⁇ : Itaconate (g/L), ⁇ : Citrate (g/L)).
  • FIG. 3 B is a diagram illustrating results of analyzing citrate and itaconate production of a WAICP VTL strain ( ⁇ : Acetate (g/L), ⁇ : Cell biomass (g DCW/L), ⁇ : Itaconate (g/L), ⁇ : Citrate (g/L)).
  • FIG. 4 is a diagram illustrating results of analyzing itaconate production of recombinant strains constructed through reverse engineering.
  • FIG. 5 is a diagram illustrating docking simulation results of PrpD and citrate of recombinant strains constructed through reverse engineering.
  • FIG. 6 is a diagram illustrating results of confirming an effect on itaconate production according to regulation of the expression level of PrpD VL according to the present disclosure.
  • an expression cassette for introducing an itaconate production pathway including a 2-methylcitrate dehydratase (PrpD) gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17; and a recombinant vector for producing itaconate including the expression cassette.
  • PrpD 2-methylcitrate dehydratase
  • the itaconate is dicarboxylic acid composed of 5 carbons, and is used as a precursor for polymer synthesis, such as plastic and latex due to structural characteristics thereof.
  • the PrpD gene is 2-methylcitrate dehydratase PrpD derived from Escherichia coli , which is known to make 2-methyl cis-aconitate by dehydrating water molecules from 2-methylcitrate.
  • the present inventors used the substrate promiscuity of the PrpD gene to construct a novel enzyme PrpD VTL that converts citrate to cis-aconitate.
  • the constructed novel enzyme PrpD VTL is represented by a nucleotide sequence set forth in SEQ ID NO: 1.
  • major mutations of the novel enzyme PrpD VTL such as PrpD V (SEQ ID NO: 7), PrpD T (SEQ ID NO: 8), PrpD L (SEQ ID NO: 9), PrpD TL (SEQ ID NO: 10), PrpD VT (SEQ ID NO: 11), and PrpD VL (SEQ ID NO: 12) were obtained.
  • the expression cassette preferably further includes a cis-aconitate decarboxylase (Cad) gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
  • Cad cis-aconitate decarboxylase
  • the cis-aconitate decarboxylase (Cad) gene is an enzyme capable of converting cis-aconitate derived from Aspergillus terreus to itaconate.
  • the Cad gene of the present disclosure is represented by the nucleotide sequence set forth in SEQ ID NO: 2.
  • a novel itaconate synthesis pathway was constructed to separate a carbon flow according to the efficiency of a new enzyme kinetic between existing competing TCA cycle and itaconate synthesis by expressing the PrpD gene and the Cad gene.
  • the novel itaconate synthesis pathway is shown in the right drawing of FIG. 1 .
  • the expression cassette may further include a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
  • the expression cassette may further include a synthetic 5′ untranslated region (UTR) represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19, preferably both synthetic 5′ UTRs represented by nucleotide sequences set forth in SEQ ID NOs: 18 and 19.
  • UTR 5′ untranslated region
  • the 5′ untranslated region is an untranslated region at 5′ end and 3′ end of mRNA, and in general, the 5′ untranslated region (5′ UTR) of mRNA performs several functions in the gene expression process, but among the functions, the largest feature is involved in regulating mRNA translation efficiency. It has been reported that a nucleotide sequence of the 5′ UTR present in an adjacent upper portion of a translation initiation codon affects the efficiency of a translation step, and the length of the 5′ UTR consists of 100 bases or more nucleotides, and the length of the 3′ UTR consist of several kilobases longer.
  • the expression cassette preferably further includes at least one promoter selected from the group consisting of SEQ ID NOs: 6 and 13 to 16.
  • the expression cassette refers to a unit cassette that includes a promoter and a gene encoding a target protein and may be expressed to produce the target protein operably linked to the downstream of the promoter.
  • Various factors capable of helping the efficient production of the target protein may be included inside or outside such an expression cassette.
  • the gene encoding the target protein may be operably linked to the downstream of a promoter sequence.
  • the gene has a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with a nucleotide sequence set forth in SEQ ID NO. corresponding to each gene, and means a sequence that exhibits substantially the same physiological activity.
  • the “% of sequence homology” with a polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a part of a polynucleotide sequence in the comparison region may include addition or deletion (i.e., gap) compared to a reference sequence (not including addition or deletion) for an optimal alignment of the two sequences.
  • the ‘operably linked’ means that the gene sequence and the promoter sequence are functionally linked to each other so that a nucleic acid sequence having the promoter activity of the present disclosure initiates and mediates the transcription of the gene encoding the target protein.
  • the operable linkage can be prepared using genetic recombination techniques known in the art, and site-specific DNA cleavage and linkage may be prepared using cleavage and linkage enzymes in the art, but are not limited thereto.
  • the ‘recombinant gene expression cassette’ can be inserted into a chromosome of a host cell and used to produce a recombinant microorganism, and for those skilled in the art to which the present disclosure pertains, it is obvious that even insertion of the recombinant gene expression cassette into the genomic chromosome of a host cell will have the same effect as the case of introducing the recombinant vector into the host cell as described above.
  • As a method of inserting the recombinant gene expression cassette into the chromosome of the host cell conventionally known genetic manipulation methods may be used.
  • a retrovirus vector an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentiviral vector, or a non-viral vector.
  • the vector refers to a genetic construct including a nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host.
  • the regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, and sequences regulating termination of transcription and translation.
  • the vector of the present disclosure is not particularly limited as long as the vector is replicable in cells, and may use any vector known in the art, for example, a plasmid, a cosmid, a phage particle, or a viral vector.
  • the recombinant vector when a coding gene of a target polypeptide to be expressed is operably linked, the recombinant vector may be used as an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell, and the recombinant vector can be expressed in a host cell.
  • the host cell may preferably be a eukaryotic cell, and expression regulatory sequences such as a promoter, a terminator, and an enhancer, sequences for membrane targeting or secretion, etc. are appropriately selected according to a type of host cell and may be variously combined depending on a purpose.
  • the expression cassette and the vector for introducing the itaconate production pathway of the present disclosure may be introduced into a microorganism to prepare a recombinant microorganism for producing the itaconate.
  • the introduced microorganism may be a wild-type E. coli W strain having resistance to acetate among E. coli , and may be a strain further including an aconitase B (AcnB) gene represented by a nucleotide sequence set forth in SEQ ID NO: 3, and/or in which an iclR gene represented by a nucleotide sequence set forth in SEQ ID NO: 4 is deleted.
  • the introduced microorganism may be a strain described in Table 1 to be described below.
  • the present disclosure provides a recombinant microorganism for producing itaconate into which a prpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • the recombinant microorganism preferably further includes a cis-aconitate decarboxylase (Cad) gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
  • Cad cis-aconitate decarboxylase
  • the recombinant microorganism may further include a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
  • the recombinant microorganism preferably further includes an aconitase B (AcnB) gene represented by a nucleotide sequence set forth in SEQ ID NO: 3.
  • the AcnB gene represented by the nucleotide sequence set forth in SEQ ID NO: 3 is an AcnB W482R variant, in which AcnB activity is reduced.
  • an iclR gene represented by a nucleotide sequence set forth in SEQ ID NO: 4 is deleted.
  • the recombinant microorganism preferably further includes at least one promoter selected from the group consisting of SEQ ID NOs: 6 and 13 to 16.
  • the recombinant microorganism may further include a synthetic 5′ untranslated region (UTR) represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19, preferably both synthetic 5′ UTRs represented by nucleotide sequences set forth in SEQ ID NOs: 18 and 19.
  • UTR 5′ untranslated region
  • the recombinant microorganism refers to a microorganism transformed with the recombinant vector of the present disclosure.
  • the ‘transformation’ means introducing a vector including the promoter according to the present disclosure or further including the gene encoding the target protein into a host cell.
  • a gene encoding the transformed target protein may be located to be inserted into the chromosome of the host cell or located outside the chromosome, as long as the gene may be expressed in the host cell.
  • one or a plurality of recombinant vectors may be introduced into the recombinant microorganism for producing the itaconate, and each of the one or the plurality of recombinant vectors may be introduced into the microorganism.
  • the recombinant vector may be sequentially introduced into the microorganism, or may also be introduced in a mutually reversed order.
  • the recombinant microorganism for producing the itaconate may be characterized to be selected from the group consisting of bacteria, yeast, and fungi, and may preferably be a microorganism of the genus Escherichia , and more preferably Escherichia coli.
  • a wild-type E. coli W strain having resistance to acetate among E. coli was used.
  • E. coli W WA Introduction of WenB W482R gene (SEQ ID NO: 3) with reduced WenB activity into E. coli W WAI Deletion of iclR gene of WA strain WAIC Introduction of recombinant expression cassette including Cad gene (SEQ ID NO: 2), Tac promoter (SEQ ID NO: 5), and synthetic 5′ UTR (SEQ ID NO: 18) into WAI strain WAICP Introduction of wild-type PrpD into WAIC strain by using recombinant expression cassette including synthetic promoter J23108 (SEQ ID NO: 6) and synthetic 5′ UTR (SEQ ID NO: 19) WAICP VTL Introduction of mutant-type PrpD VTL into WAIC strain by using recombinant expression cassette including synthetic promoter J23108 (SEQ ID NO: 6) and synthetic 5′ UTR (SEQ ID NO: 19) WAICP V Overexpression by introducing recombinant plasmid including PrpD V (SEQ ID NO: 7), J
  • the present disclosure provides a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • any medium and other culture conditions used for culturing the microorganism of the present disclosure may be used with any medium used for culturing conventional microorganisms of the genus Escherichia , but need to suitably satisfy the requirements of the microorganism of the present disclosure.
  • the microorganism of the present disclosure is cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. under aerobic conditions while controlling temperature, pH, and the like.
  • the medium may include glucose, pyruvate, etc. as a carbon source.
  • a carbon source As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used, and in addition, amino acids, vitamins, appropriate precursors, and the like may be included. These media or precursors may be added to a culture solution in a batch or continuous manner.
  • a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid is added to the culture solution by a proper method to adjust the pH of the culture solution.
  • an anti-foaming agent such as fatty acid polyglycol ester.
  • oxygen or oxygen-containing gases may be injected into the culture solution, and in order to maintain anaerobic and aerobic states, gases are not injected or nitrogen, hydrogen, or carbon dioxide gas may be injected.
  • the temperature of the culture solution may be set usually 27° C. to 37° C., preferably 30° C. to 35° C.
  • the culturing period may be continued until a desired production amount of a useful substance is obtained, preferably for 10 to 100 hours.
  • the method may further include purifying or recovering the itaconate produced in the culturing step of the present disclosure, and a method for recovering the itaconate from the microorganism or culture solution may be used with methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but is not limited to these examples.
  • the recovering step may include a purification process, and those skilled in the art may select and utilize several well-known purification processes as needed.
  • the recombinant microorganism for producing the itaconate of the present disclosure and the method for producing the itaconate using the same, it is possible to increase a production capacity of itaconate through the establishment of a novel synthesis pathway of accumulating cis-aconitate, a precursor of itaconate and an intermediate of a TCA cycle, in the microorganism, and separating the existing competing TCA cycle and itaconate synthesis.
  • an AcnB W482R gene (SEQ ID NO: 3) with reduced AcnB activity was introduced into wild-type E. coli W to construct a WA strain.
  • a WAI strain from which an iclR gene (SEQ ID NO: 4) was deleted was constructed in the constructed WA strain.
  • a WAIC strain was constructed by introducing a recombinant expression cassette including a Cad gene (SEQ ID NO: 2), a Tac promoter (SEQ ID NO: 5), and a synthetic 5′ UTR (SEQ ID NO: 18) into the WAI strain.
  • a WCI strain was prepared in which a Cad gene recombination expression cassette was introduced into wild-type E. coli W while the iclR gene was deleted.
  • PrpD VTL SEQ ID NO: 1 of converting citrate to cis-aconitate, was constructed by using the substrate promiscuity of a 2-methylcitrate dehydratase (PrpD) gene.
  • PrpD 2-methylcitrate dehydratase
  • the novel enzyme PrpD VTL was an enzyme in which tryptophan No. 110, glycine No. 111, and isoleucine No. 331 based on wild-type PrpD (Accession No. NC_000913) were mutated to valine, threonine, and leucine, respectively.
  • a novel itaconate synthesis pathway was constructed to separate a carbon flow according to the efficiency of a new enzyme kinetic between existing competing TCA cycle and itaconate synthesis by expressing the PrpD VTL and the Cad gene (SEQ ID NO: 2).
  • a schematic diagram of enzyme kinetic-based compartmentalization according to a reaction of the novel enzyme was shown in FIG. 1 .
  • a comparison of reaction substrates and reaction mechanisms of 2-methylcitrate dehydratase and aconitase was shown in FIG. 2 .
  • Wild-type PrpD and screened mutant-type PrpD VTL were introduced into a WAIC strain by using a recombinant expression cassette including a synthetic promoter J23108 (SEQ ID NO: 6) and a synthetic 5′ UTR (SEQ ID NO: 19), respectively, to construct WAICP and WAICP VTL strains.
  • the constructed WAICP and WAICP VTL strains were cultured. Specifically, each of the two constructed strains was cultured on a solid LB agar plate to obtain individual colonies. The obtained individual colonies were cultured for about 12 hours in a flask under conditions of 30° C. and 200 rpm. The cultured strains were inoculated at a 1/100 dilution into 20 mL of a production medium contained in a 400 mL round flask, added with IPTG at a concentration of 0.1 mM, and then cultured under conditions of 30° C. and 200 rpm.
  • the strains were inoculated into 20 mL of a production medium contained in a 300 mL round flask to become an OD 600 value of 0.05, and added with IPTG at a concentration of 0.1 mM. Thereafter, the strain was cultured under conditions of 30° C. and 200 rpm.
  • the production medium used in the example was based on an M9 medium, and additionally added with 10 g/L of acetate and 2 g/L of yeast extract. At intervals of 12 hours of culture, 1 mL of the culture solution was separated from the culture cells by centrifugation. A supernatant was taken from the separated culture solution and quantitatively analyzed by HPLC.
  • the HPLC analysis was performed using an Aminex HPX-87H column as a stationary phase and an aqueous 5 mM sulfuric acid solution as a mobile phase at a mobile phase rate of 0.6 mL per minute, and a Shodex RI-101 instrument was used for detection.
  • the results of citrate and itaconate production of the constructed WAICP and WAICP VTL strains were shown in FIGS. 3 A and 3 B , respectively.
  • Example 3 it was confirmed which residue among mutated residues of the screened PrpD-based novel cis-aconitate synthesis enzyme had a major effect on increasing the reactivity to citrate.
  • reverse engineering was performed by mutating one or two of three mutated amino acid sequences. PrpD mutants for reverse engineering were as follows.
  • PrpD V (SEQ ID NO: 7)
  • PrpD L (SEQ ID NO: 9)
  • a PrpD variant prepared through the reverse engineering was introduced into a recombinant plasmid containing a J23108 promoter (SEQ ID NO: 6) and a synthetic 5′ UTR (SEQ ID NO: 19) to be overexpressed.
  • a recombinant plasmid containing a J23108 promoter SEQ ID NO: 6
  • a synthetic 5′ UTR SEQ ID NO: 19
  • recombinant strains were constructed by introducing recombinant plasmids of wild-type PrpD, a mutant type of PrpD, a screened novel cis-aconitate synthesis enzyme, and mutant types of PrpD produced through reverse engineering.
  • the constructed recombinant strains were as follows.
  • Example 2 Each of the 8 recombinant strains constructed in Example 2 and Example 3 was cultured to produce itaconate, and the produced itaconate was quantified. The production and quantification of itaconate were performed in the same manner as in Example 2. The production results of itaconate of the 8 constructed recombinant strains were shown in FIG. 4 .
  • Example 4 the itaconate production capacity was analyzed according to a control of the expression level of the WAICP VL strain, which had a significantly higher itaconate production capacity than the WAICP VTL strain obtained through reverse engineering in Example 3. That is, it was confirmed whether a carbon flow toward itaconate could be separated from the existing TCA cycle according to the regulation of the expression level of PrpD VL .
  • the constructed recombinant strains produced and quantified itaconate in the same manner as in Example 2 above.
  • the results of analyzing the itaconate production of the constructed recombinant strain were shown in FIG. 6 .
  • the production of itaconate in the recombinant strain WAICP100 VL for 48 hours was about 1.45-fold increased compared to the recombinant strain WAICP114 VL . Accordingly, it was confirmed that the carbon flow toward itaconate was separated according to the expression level of the novel cis-aconitate synthesis enzyme. This means that the recombinant strain can separate the carbon flow from the existing TCA cycle according to the reactivity of the newly constructed cis-aconitate synthesis enzyme.
  • the present inventors have improved the novel cis-aconitate synthesis enzyme and found that the new carbon flow to itaconate of the recombinant microorganisms introduced with the novel cis-aconitate synthesis enzyme was separated from the existing TCA cycle based on the activity of the enzyme, and finally the productivity of itaconate was increased. Therefore, the recombinant microorganism of the present disclosure can be variously used in the field of itaconate production.

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Abstract

Provided is a novel cis-aconitate synthesis enzyme, more particularly, a recombinant microorganism for producing itaconate including a cis-aconitate synthesis enzyme variant. According to the present disclosure, it was confirmed that the production and yield of itaconate were significantly increased in the recombinant microorganism for producing itaconate into which the novel cis-aconitate synthesis enzyme was introduced. In addition, it was confirmed that in the recombinant microorganism for producing itaconate of the present disclosure, a new carbon flow to itaconate was separated from the existing TCA cycle based on the activity of the corresponding enzyme. Accordingly, the novel aconitate synthesis enzyme of the present disclosure and the recombinant microorganism introduced with the aconitate synthesis enzyme can increase the economic feasibility of itaconate, and thus can be used in various industrial fields, such as synthetic resins, latexes, and food additives in which itaconate is used.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is based on and claims priority from Korean Patent Application No. 10-2022-0042345 filed on Apr. 5, 2022 and Application No. 10-2022-01895874 filed on Dec. 29, 2022 with the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
    • REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
  • The contents of the electronic sequence listing (JPG20232077US_SEQ.xml; Size: 37 K bytes; and Date of Creation: Mar. 28, 2023) is herein incorporated by reference in its entirety.
    • TECHNICAL FIELD
  • The present disclosure relates to a novel cis-aconitate synthesis enzyme, and more particularly, to a recombinant microorganism for producing itaconate including a cis-aconitate synthesis enzyme variant.
    • BACKGROUND
  • Itaconate (itaconic acid), dicarboxylic acid composed of 5 carbons, is a material that may be used as precursors of various polymer materials such as latex and plastics, drug transport, and water purification based on structural characteristics of the material. Due to great industrial potential, the itaconate was selected as one of 12 bio-based platform chemicals selected by the United States Department of Energy in 2004.
  • In the case of itaconate, there is a case of Aspergillus terreus, which produces naturally the itaconate, but in Aspergillus terreus, genetic manipulation is difficult due to relatively insufficient genetic manipulation tools, and there are still limitations in mass production due to difficult fermentation conditions due to the characteristics of filamentous fungi. As an alternative, a study to establish a recombinant microorganism for efficient production of itaconate in the form of introducing cis-aconitate decarboxylase (Cad) derived from Aspergillus terreus using Escherichia coli, one of the industrial strains has been actively conducted.
  • However, the production of itaconate through recombinant E. coli has been limited in that enzyme expression and activity are reduced when heterologous expression occurs, and that cis-aconitate, a precursor of itaconate and an intermediate in a TCA cycle, is hardly accumulated in E. coli.
    • SUMMARY
  • Accordingly, in order to solve the problems of the related art as described above, the present inventors have developed a novel cis-aconitate synthesis enzyme through an evolutionary design rather than existing enzymes with respect to the synthesis of cis-aconitate, an intermediate in the TCA cycle to intend to increase the efficiency of itaconate synthesis by separating existing competing TCA cycle and itaconate synthesis according to the activity of the novel enzyme. In addition, the present inventors confirmed that a recombinant microorganism introduced with the enzyme responsible for the biosynthetic reaction had excellent itaconate production capacity and then completed the present disclosure.
  • The present disclosure has been made in an effort to provide an expression cassette for introducing an itaconate production pathway including a 2-methylcitrate dehydratase (PrpD) gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17.
  • The present disclosure has also been made in an effort to provide a recombinant vector for producing an itaconate production pathway including the expression cassette.
  • The present disclosure has also been made in an effort to provide a recombinant microorganism for producing itaconate into which a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • The present disclosure has also been made in an effort to provide a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • An embodiment of the present disclosure provides an expression cassette for introducing an itaconate production pathway including a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17.
  • Another embodiment of the present disclosure provides a recombinant vector for producing itaconate including the expression cassette for introducing the itaconate production pathway.
  • Yet another embodiment of the present disclosure provides a recombinant microorganism for producing itaconate into which a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • Still another embodiment of the present disclosure provides a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • According to the embodiment of the present disclosure, it was confirmed that the production and yield of itaconate were significantly increased in the recombinant microorganism for producing itaconate into which the novel cis-aconitate synthesis enzyme was introduced. In addition, it was confirmed that in the recombinant microorganism for producing itaconate of the present disclosure, a new carbon flow to itaconate was separated from the existing TCA cycle based on the activity of the corresponding enzyme. Accordingly, the novel aconitate synthesis enzyme of the present disclosure and the recombinant microorganism introduced with the aconitate synthesis enzyme can increase the economic feasibility of itaconate, and thus can be used in various industrial fields, such as synthetic resins, latexes, and food additives in which itaconate is used.
  • The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a diagram illustrating an itaconate production pathway through introduction of a novel cis-aconitate synthesis enzyme in E. coli according to the present disclosure and a carbon flow compartmentalization according to the activity of the enzyme (Left: existing itaconate production pathway, Right: novel itaconate production pathway according to the present disclosure).
  • FIG. 2 is a diagram illustrating a comparison of a reaction mechanism and a reaction substrate of 2-methylstarate dehydratase (PrpD) and aconitase, which are wild types of the novel cis-aconitate synthesis enzyme according to the present disclosure.
  • FIG. 3A is a diagram illustrating results of analyzing citrate and itaconate production of a WAICP strain according to the present disclosure (▴: Acetate (g/L), ●: Cell biomass (g DCW/L), ▪: Itaconate (g/L), ▾: Citrate (g/L)).
  • FIG. 3B is a diagram illustrating results of analyzing citrate and itaconate production of a WAICPVTL strain (▴: Acetate (g/L), ●: Cell biomass (g DCW/L), ▪: Itaconate (g/L), ▾: Citrate (g/L)).
  • FIG. 4 is a diagram illustrating results of analyzing itaconate production of recombinant strains constructed through reverse engineering.
  • FIG. 5 is a diagram illustrating docking simulation results of PrpD and citrate of recombinant strains constructed through reverse engineering.
  • FIG. 6 is a diagram illustrating results of confirming an effect on itaconate production according to regulation of the expression level of PrpDVL according to the present disclosure.
    •  DETAILED DESCRIPTION
  • Hereinafter, the present disclosure will be described in detail.
  • According to an aspect of the present disclosure, there are provided an expression cassette for introducing an itaconate production pathway including a 2-methylcitrate dehydratase (PrpD) gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17; and a recombinant vector for producing itaconate including the expression cassette.
  • In the present disclosure, the itaconate is dicarboxylic acid composed of 5 carbons, and is used as a precursor for polymer synthesis, such as plastic and latex due to structural characteristics thereof.
  • In the present disclosure, the PrpD gene is 2-methylcitrate dehydratase PrpD derived from Escherichia coli, which is known to make 2-methyl cis-aconitate by dehydrating water molecules from 2-methylcitrate.
  • The present inventors used the substrate promiscuity of the PrpD gene to construct a novel enzyme PrpDVTL that converts citrate to cis-aconitate. The constructed novel enzyme PrpDVTL is represented by a nucleotide sequence set forth in SEQ ID NO: 1. In addition, through reverse engineering, major mutations of the novel enzyme PrpDVTL, such as PrpDV (SEQ ID NO: 7), PrpDT (SEQ ID NO: 8), PrpDL (SEQ ID NO: 9), PrpDTL (SEQ ID NO: 10), PrpDVT (SEQ ID NO: 11), and PrpDVL (SEQ ID NO: 12) were obtained.
  • In an embodiment of the present disclosure, the expression cassette preferably further includes a cis-aconitate decarboxylase (Cad) gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
  • In the present disclosure, the cis-aconitate decarboxylase (Cad) gene is an enzyme capable of converting cis-aconitate derived from Aspergillus terreus to itaconate. The Cad gene of the present disclosure is represented by the nucleotide sequence set forth in SEQ ID NO: 2.
  • In the present disclosure, a novel itaconate synthesis pathway was constructed to separate a carbon flow according to the efficiency of a new enzyme kinetic between existing competing TCA cycle and itaconate synthesis by expressing the PrpD gene and the Cad gene. The novel itaconate synthesis pathway is shown in the right drawing of FIG. 1 .
  • In a preferred embodiment of the present disclosure, the expression cassette may further include a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
  • In an embodiment of the present disclosure, the expression cassette may further include a synthetic 5′ untranslated region (UTR) represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19, preferably both synthetic 5′ UTRs represented by nucleotide sequences set forth in SEQ ID NOs: 18 and 19.
  • In the present disclosure, the 5′ untranslated region (UTR) is an untranslated region at 5′ end and 3′ end of mRNA, and in general, the 5′ untranslated region (5′ UTR) of mRNA performs several functions in the gene expression process, but among the functions, the largest feature is involved in regulating mRNA translation efficiency. It has been reported that a nucleotide sequence of the 5′ UTR present in an adjacent upper portion of a translation initiation codon affects the efficiency of a translation step, and the length of the 5′ UTR consists of 100 bases or more nucleotides, and the length of the 3′ UTR consist of several kilobases longer. In addition, it has been reported results of studies on sequences belonging to the 5′ UTR, which may be referred to as ribosome binding site sequences in eukaryotes, not at a fixed position such as a Shine-Dalgarno sequence, which was known as a ribosome binding site sequence located in the 5′ UTR in prokaryotes.
  • In an embodiment of the present disclosure, the expression cassette preferably further includes at least one promoter selected from the group consisting of SEQ ID NOs: 6 and 13 to 16.
  • In the present disclosure, the expression cassette refers to a unit cassette that includes a promoter and a gene encoding a target protein and may be expressed to produce the target protein operably linked to the downstream of the promoter. Various factors capable of helping the efficient production of the target protein may be included inside or outside such an expression cassette. Specifically, in the target protein expression cassette, specifically, the gene encoding the target protein may be operably linked to the downstream of a promoter sequence.
  • In addition, variants of the gene are also included within the scope of the present disclosure. Specifically, the gene has a sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with a nucleotide sequence set forth in SEQ ID NO. corresponding to each gene, and means a sequence that exhibits substantially the same physiological activity. The “% of sequence homology” with a polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a part of a polynucleotide sequence in the comparison region may include addition or deletion (i.e., gap) compared to a reference sequence (not including addition or deletion) for an optimal alignment of the two sequences.
  • The ‘operably linked’ means that the gene sequence and the promoter sequence are functionally linked to each other so that a nucleic acid sequence having the promoter activity of the present disclosure initiates and mediates the transcription of the gene encoding the target protein. The operable linkage can be prepared using genetic recombination techniques known in the art, and site-specific DNA cleavage and linkage may be prepared using cleavage and linkage enzymes in the art, but are not limited thereto. That is, the ‘recombinant gene expression cassette’ can be inserted into a chromosome of a host cell and used to produce a recombinant microorganism, and for those skilled in the art to which the present disclosure pertains, it is obvious that even insertion of the recombinant gene expression cassette into the genomic chromosome of a host cell will have the same effect as the case of introducing the recombinant vector into the host cell as described above. As a method of inserting the recombinant gene expression cassette into the chromosome of the host cell, conventionally known genetic manipulation methods may be used. As an example, there is a method using a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentiviral vector, or a non-viral vector.
  • In the present disclosure, the vector refers to a genetic construct including a nucleotide sequence of a gene operably linked to a suitable regulatory sequence so as to express a target gene in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, and sequences regulating termination of transcription and translation. The vector of the present disclosure is not particularly limited as long as the vector is replicable in cells, and may use any vector known in the art, for example, a plasmid, a cosmid, a phage particle, or a viral vector.
  • In the present disclosure, when a coding gene of a target polypeptide to be expressed is operably linked, the recombinant vector may be used as an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell, and the recombinant vector can be expressed in a host cell. The host cell may preferably be a eukaryotic cell, and expression regulatory sequences such as a promoter, a terminator, and an enhancer, sequences for membrane targeting or secretion, etc. are appropriately selected according to a type of host cell and may be variously combined depending on a purpose.
  • The expression cassette and the vector for introducing the itaconate production pathway of the present disclosure may be introduced into a microorganism to prepare a recombinant microorganism for producing the itaconate. At this time, the introduced microorganism may be a wild-type E. coli W strain having resistance to acetate among E. coli, and may be a strain further including an aconitase B (AcnB) gene represented by a nucleotide sequence set forth in SEQ ID NO: 3, and/or in which an iclR gene represented by a nucleotide sequence set forth in SEQ ID NO: 4 is deleted. As a specific embodiment, the introduced microorganism may be a strain described in Table 1 to be described below.
  • According to yet another aspect of the present disclosure, the present disclosure provides a recombinant microorganism for producing itaconate into which a prpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
  • In an embodiment of the present disclosure, the recombinant microorganism preferably further includes a cis-aconitate decarboxylase (Cad) gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
  • In a preferred embodiment of the present disclosure, the recombinant microorganism may further include a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
  • In an embodiment of the present disclosure, the recombinant microorganism preferably further includes an aconitase B (AcnB) gene represented by a nucleotide sequence set forth in SEQ ID NO: 3. The AcnB gene represented by the nucleotide sequence set forth in SEQ ID NO: 3 is an AcnB W482R variant, in which AcnB activity is reduced.
  • In an embodiment of the present disclosure, in the recombinant microorganism, preferably, an iclR gene represented by a nucleotide sequence set forth in SEQ ID NO: 4 is deleted.
  • In an embodiment of the present disclosure, the recombinant microorganism preferably further includes at least one promoter selected from the group consisting of SEQ ID NOs: 6 and 13 to 16.
  • In an embodiment of the present disclosure, the recombinant microorganism may further include a synthetic 5′ untranslated region (UTR) represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19, preferably both synthetic 5′ UTRs represented by nucleotide sequences set forth in SEQ ID NOs: 18 and 19.
  • In the present disclosure, the recombinant microorganism refers to a microorganism transformed with the recombinant vector of the present disclosure. In the present disclosure, the ‘transformation’ means introducing a vector including the promoter according to the present disclosure or further including the gene encoding the target protein into a host cell. In addition, a gene encoding the transformed target protein may be located to be inserted into the chromosome of the host cell or located outside the chromosome, as long as the gene may be expressed in the host cell.
  • In the present disclosure, one or a plurality of recombinant vectors may be introduced into the recombinant microorganism for producing the itaconate, and each of the one or the plurality of recombinant vectors may be introduced into the microorganism. In addition, the recombinant vector may be sequentially introduced into the microorganism, or may also be introduced in a mutually reversed order.
  • In an embodiment of the present disclosure, the recombinant microorganism for producing the itaconate may be characterized to be selected from the group consisting of bacteria, yeast, and fungi, and may preferably be a microorganism of the genus Escherichia, and more preferably Escherichia coli.
  • In an embodiment of the present disclosure, a wild-type E. coli W strain having resistance to acetate among E. coli was used.
  • Recombinant microorganisms constructed in the present disclosure are shown in Table 1 below.
  • TABLE 1
    Strain name Characteristics
    E. coli W —
    WA Introduction of WenB W482R gene (SEQ ID NO: 3) with
    reduced WenB activity into E. coli W
    WAI Deletion of iclR gene of WA strain
    WAIC Introduction of recombinant expression cassette including Cad
    gene (SEQ ID NO: 2), Tac promoter (SEQ ID NO: 5), and
    synthetic 5′ UTR (SEQ ID NO: 18) into WAI strain
    WAICP Introduction of wild-type PrpD into WAIC strain by using
    recombinant expression cassette including synthetic promoter
    J23108 (SEQ ID NO: 6) and synthetic 5′ UTR (SEQ ID NO:
    19)
    WAICPVTL Introduction of mutant-type PrpDVTL into WAIC strain by
    using recombinant expression cassette including synthetic
    promoter J23108 (SEQ ID NO: 6) and synthetic 5′ UTR (SEQ
    ID NO: 19)
    WAICPV Overexpression by introducing recombinant plasmid including
    PrpDV (SEQ ID NO: 7), J23108 promoter (SEQ ID NO: 6),
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICPT Overexpression by introducing recombinant plasmid including
    PrpDT (SEQ ID NO: 8), J23108 promoter (SEQ ID NO: 6)
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICPL Overexpression by introducing recombinant plasmid including
    PrpDL (SEQ ID NO: 9), J23108 promoter (SEQ ID NO: 6),
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICPTL Overexpression by introducing recombinant plasmid including
    PrpDTL (SEQ ID NO: 10), J23108 promoter (SEQ ID NO: 6),
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICPVT Overexpression by introducing recombinant plasmid including
    PrpDVT (SEQ ID NO: 11), J23108 promoter (SEQ ID NO: 6),
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICPVL Overexpression by introducing recombinant plasmid including
    PrpVL (SEQ ID NO: 12), J23108 promoter (SEQ ID NO: 6),
    and synthetic 5′ UTR (SEQ ID NO: 19) into WAIC strain
    WAICP115VL Application of J23115 promoter (SEQ ID NO: 13) and
    synthetic 5′ UTR (SEQ ID NO: 19) to WAICPVL strain
    WAICP114VL Application of J23114 promoter (SEQ ID NO: 14) and
    synthetic 5′ UTR (SEQ ID NO: 19) to WAICPVL strain
    WAICP110VL Application of J23100 promoter (SEQ ID NO: 15) and
    synthetic 5′ UTR (SEQ ID NO: 19) to WAICPVL strain
    WAICP100VL Application of J23100 promoter (SEQ ID NO: 16) and
    synthetic 5′ UTR (SEQ ID NO: 19) to WAICPVL strain
    WCI (control) Introduction of Cad gene recombination expression cassette
    with deleted iclR gene into E. coli W
  • According to yet another aspect of the present disclosure, the present disclosure provides a method for producing itaconate including culturing a recombinant microorganism for producing itaconate.
  • Any medium and other culture conditions used for culturing the microorganism of the present disclosure may be used with any medium used for culturing conventional microorganisms of the genus Escherichia, but need to suitably satisfy the requirements of the microorganism of the present disclosure. Preferably, the microorganism of the present disclosure is cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. under aerobic conditions while controlling temperature, pH, and the like.
  • In a preferred embodiment of the present disclosure, the medium may include glucose, pyruvate, etc. as a carbon source. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used, and in addition, amino acids, vitamins, appropriate precursors, and the like may be included. These media or precursors may be added to a culture solution in a batch or continuous manner.
  • During the culture, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid is added to the culture solution by a proper method to adjust the pH of the culture solution. In addition, during the culture, production of bubbles may be inhibited by using an anti-foaming agent such as fatty acid polyglycol ester. Further, in order to maintain an aerobic state of the culture solution, oxygen or oxygen-containing gases may be injected into the culture solution, and in order to maintain anaerobic and aerobic states, gases are not injected or nitrogen, hydrogen, or carbon dioxide gas may be injected.
  • The temperature of the culture solution may be set usually 27° C. to 37° C., preferably 30° C. to 35° C. The culturing period may be continued until a desired production amount of a useful substance is obtained, preferably for 10 to 100 hours.
  • The method may further include purifying or recovering the itaconate produced in the culturing step of the present disclosure, and a method for recovering the itaconate from the microorganism or culture solution may be used with methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but is not limited to these examples.
  • The recovering step may include a purification process, and those skilled in the art may select and utilize several well-known purification processes as needed.
  • According to the recombinant microorganism for producing the itaconate of the present disclosure and the method for producing the itaconate using the same, it is possible to increase a production capacity of itaconate through the establishment of a novel synthesis pathway of accumulating cis-aconitate, a precursor of itaconate and an intermediate of a TCA cycle, in the microorganism, and separating the existing competing TCA cycle and itaconate synthesis.
  • Duplicated contents are omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have the meanings commonly used in the art to which the present disclosure pertains.
  • Hereinafter, the present disclosure will be described in more detail through Examples. These Examples are just illustrative of the present disclosure, and it will be apparent to those skilled in the art that it is not interpreted that the scope of the present disclosure is limited to these Examples.
    • Example 1. Construction of Strain with Increased Intracellular Citrate Concentration for Improvement of Novel Cis-Aconitate Synthesis Enzyme
  • First, an AcnB W482R gene (SEQ ID NO: 3) with reduced AcnB activity was introduced into wild-type E. coli W to construct a WA strain. A WAI strain from which an iclR gene (SEQ ID NO: 4) was deleted was constructed in the constructed WA strain. A WAIC strain was constructed by introducing a recombinant expression cassette including a Cad gene (SEQ ID NO: 2), a Tac promoter (SEQ ID NO: 5), and a synthetic 5′ UTR (SEQ ID NO: 18) into the WAI strain.
  • In addition, as a control, a WCI strain was prepared in which a Cad gene recombination expression cassette was introduced into wild-type E. coli W while the iclR gene was deleted.
    • Example 2. Confirmation of Itaconate Production of Recombinant Microorganism Introduced with Novel Cis-Aconitate Synthesis Enzyme
  • A novel enzyme PrpDVTL (SEQ ID NO: 1) of converting citrate to cis-aconitate, was constructed by using the substrate promiscuity of a 2-methylcitrate dehydratase (PrpD) gene. The novel enzyme PrpDVTL was an enzyme in which tryptophan No. 110, glycine No. 111, and isoleucine No. 331 based on wild-type PrpD (Accession No. NC_000913) were mutated to valine, threonine, and leucine, respectively. Specifically, a novel itaconate synthesis pathway was constructed to separate a carbon flow according to the efficiency of a new enzyme kinetic between existing competing TCA cycle and itaconate synthesis by expressing the PrpDVTL and the Cad gene (SEQ ID NO: 2). A schematic diagram of enzyme kinetic-based compartmentalization according to a reaction of the novel enzyme was shown in FIG. 1 . In addition, a comparison of reaction substrates and reaction mechanisms of 2-methylcitrate dehydratase and aconitase was shown in FIG. 2 .
  • Wild-type PrpD and screened mutant-type PrpDVTL were introduced into a WAIC strain by using a recombinant expression cassette including a synthetic promoter J23108 (SEQ ID NO: 6) and a synthetic 5′ UTR (SEQ ID NO: 19), respectively, to construct WAICP and WAICPVTL strains.
  • In order to confirm that itaconate production was improved by the novel cis-aconitate synthesis enzyme, the constructed WAICP and WAICPVTL strains were cultured. Specifically, each of the two constructed strains was cultured on a solid LB agar plate to obtain individual colonies. The obtained individual colonies were cultured for about 12 hours in a flask under conditions of 30° C. and 200 rpm. The cultured strains were inoculated at a 1/100 dilution into 20 mL of a production medium contained in a 400 mL round flask, added with IPTG at a concentration of 0.1 mM, and then cultured under conditions of 30° C. and 200 rpm. Then, when an OD600 value reached 1 to 2, the strains were inoculated into 20 mL of a production medium contained in a 300 mL round flask to become an OD600 value of 0.05, and added with IPTG at a concentration of 0.1 mM. Thereafter, the strain was cultured under conditions of 30° C. and 200 rpm. The production medium used in the example was based on an M9 medium, and additionally added with 10 g/L of acetate and 2 g/L of yeast extract. At intervals of 12 hours of culture, 1 mL of the culture solution was separated from the culture cells by centrifugation. A supernatant was taken from the separated culture solution and quantitatively analyzed by HPLC. The HPLC analysis was performed using an Aminex HPX-87H column as a stationary phase and an aqueous 5 mM sulfuric acid solution as a mobile phase at a mobile phase rate of 0.6 mL per minute, and a Shodex RI-101 instrument was used for detection. The results of citrate and itaconate production of the constructed WAICP and WAICPVTL strains were shown in FIGS. 3A and 3B, respectively.
  • As shown in FIGS. 3A and 3B, it was confirmed that the citrate production of the WAICPVTL strain was significantly increased compared to that of the WAICP strain.
    • Example 3. Identification of Key Mutant Residues Through Reverse Engineering and Preparation of Recombinant Microbial Strains with Further Improved Itaconate Production Capacity
  • In Example 3, it was confirmed which residue among mutated residues of the screened PrpD-based novel cis-aconitate synthesis enzyme had a major effect on increasing the reactivity to citrate. To this end, reverse engineering was performed by mutating one or two of three mutated amino acid sequences. PrpD mutants for reverse engineering were as follows.
  • PrpDV (SEQ ID NO: 7)
  • PrpDT (SEQ ID NO: 8)
  • PrpDL (SEQ ID NO: 9)
  • PrpDTL (SEQ ID NO: 10)
  • PrpDVT (SEQ ID NO: 11)
  • PrpDVL (SEQ ID NO: 12)
  • In addition, a PrpD variant prepared through the reverse engineering was introduced into a recombinant plasmid containing a J23108 promoter (SEQ ID NO: 6) and a synthetic 5′ UTR (SEQ ID NO: 19) to be overexpressed. Based on the WAIC strain with an increased intracellular concentration of citrate while the production of itaconate was confirmed in Example 1, recombinant strains were constructed by introducing recombinant plasmids of wild-type PrpD, a mutant type of PrpD, a screened novel cis-aconitate synthesis enzyme, and mutant types of PrpD produced through reverse engineering. The constructed recombinant strains were as follows.
      • WAICP: Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and wild-type PrpD while IclR was deleted
      • WAICPVTL : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and screened novel cis-aconitate synthesis enzyme PrpDVTL while IclR was deleted
      • WAICPV : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and PrpDV prepared through reverse engineering while IclR was deleted
      • WAICPT : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and PrpDT prepared through reverse engineering while IclR was deleted
      • WAICPL : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and PrpDL prepared through reverse engineering while IclR was deleted
      • WAICPTL : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and PrpDTL prepared through reverse engineering while IclR was deleted
      • WAICPVT : Escherichia coli W having AcnB W482R with low AcnB activity, and introduced with Cad and PrpDVT prepared through reverse engineering while IclR was deleted
      • WAICPVL : Escherichia coli W having AcnB W482R with low AcnB activity, and expressed with Cad and PrpDVL prepared through reverse engineering by J23108 promoter and synthetic 5′ UTR while IclR was deleted
  • Each of the 8 recombinant strains constructed in Example 2 and Example 3 was cultured to produce itaconate, and the produced itaconate was quantified. The production and quantification of itaconate were performed in the same manner as in Example 2. The production results of itaconate of the 8 constructed recombinant strains were shown in FIG. 4 .
  • As shown in FIG. 4 , the itaconate production strains WAICPVTL, WAICPVT, and WAICPVL introduced with a mutant of PrpD, which was a screened novel citrate synthesis enzyme, were significantly increased in itaconate productivity compared to the previous strains.
  • In order to analyze the interaction between citrate and residues near an active site of the enzyme, docking simulations of wild-type PrpD and improved novel PrpD variants were performed. The docking simulation analysis results were shown in FIG. 5 .
  • As shown in FIG. 5 , it was confirmed through docking simulation that the reactivity of PrpD to citrate was changed.
    • Example 4. Confirmation of Difference in Itaconate Production Through Regulation of Expression Level of Novel Citrate Synthesis Enzyme and Demonstration of Carbon Flow Compartmentalization Based on Enzyme Kinetic
  • In Example 4, the itaconate production capacity was analyzed according to a control of the expression level of the WAICPVL strain, which had a significantly higher itaconate production capacity than the WAICPVTL strain obtained through reverse engineering in Example 3. That is, it was confirmed whether a carbon flow toward itaconate could be separated from the existing TCA cycle according to the regulation of the expression level of PrpDVL.
  • As a result, an unnatural reaction for newly synthesizing cis-aconitate was constructed through a novel PrpD-based aconitate synthesis enzyme without the existing TCA cycle for temporarily synthesizing cis-aconitate. The carbon flow through the reaction could be changed according to the regulation of the expression level of the novel aconitate synthesis enzyme. This means that carbon flow can be compartmentalized in the existing TCA cycle based on the activity of the novel enzyme, so that the expression level was regulated by varying a synthetic promoter of PrpDVL of a strain with the highest itaconate concentration. Recombinant strains constructed with different synthetic promoters of PrpDVL were as follows.
      • WAICP115VL : Escherichia coli W having AcnB W482R with low AcnB activity, and expressing Cad and PrpDVL prepared through reverse engineering by J23115 promoter (SEQ ID NO: 13) and synthetic 5′ UTR (SEQ ID NO: 19) while IclR was deleted
      • WAICP114VL : Escherichia coli W having AcnB W482R with low AcnB activity, and expressing Cad and PrpDVL prepared through reverse engineering by J23114 promoter (SEQ ID NO: 14) and synthetic 5′ UTR (SEQ ID NO: 19) while IclR was deleted
      • WAICP110VL : Escherichia coli W having AcnB W482R with low AcnB activity, and expressing Cad and PrpDVL prepared through reverse engineering by J23110 promoter (SEQ ID NO: 15) and synthetic 5′ UTR (SEQ ID NO: 19) while IclR was deleted
      • WAICP100VL : Escherichia coli W having AcnB W482R with low AcnB activity, and expressing Cad and PrpDVL prepared through reverse engineering by J23100 promoter (SEQ ID NO: 16) and synthetic 5′ UTR (SEQ ID NO: 19) while IclR was deleted
  • The constructed recombinant strains produced and quantified itaconate in the same manner as in Example 2 above. The results of analyzing the itaconate production of the constructed recombinant strain were shown in FIG. 6 .
  • As shown in FIG. 6 , the production of itaconate in the recombinant strain WAICP100VL for 48 hours was about 1.45-fold increased compared to the recombinant strain WAICP114VL. Accordingly, it was confirmed that the carbon flow toward itaconate was separated according to the expression level of the novel cis-aconitate synthesis enzyme. This means that the recombinant strain can separate the carbon flow from the existing TCA cycle according to the reactivity of the newly constructed cis-aconitate synthesis enzyme.
  • All sequences of genes used in Examples of the present disclosure are shown in Table 2.
  • TABLE 2
    SEQ ID
    NO: Name Sequence
    1 PrpDVTL ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGgttactCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCct
    aATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    2 Cad ATGACCAAACAGAGCGCAGATAGCAATGCAAA
    AAGCGGTGTTACCAGCGAAATTTGTCATTGGG
    CAAGCAATCTGGCAACCGATGATATTCCGAGT
    GATGTTCTGGAACGTGCCAAATATCTGATTCT
    GGATGGTATTGCATGTGCATGGGTTGGTGCAC
    GTGTTCCGTGGTCAGAAAAATATGTTCAGGCA
    ACCATGAGCTTTGAACCGCCTGGTGCATGTCG
    TGTTATTGGTTATGGCCAGAAACTGGGTCCGG
    TTGCAGCAGCAATGACCAATAGCGCATTTATT
    CAGGCCACCGAACTGGATGATTATCATAGCGA
    AGCACCGCTGCATAGCGCAAGCATTGTTCTGC
    CTGCAGTTTTTGCAGCAAGCGAAGTTCTGGCA
    GAACAGGGTAAAACCATTAGCGGTATTGATGT
    TATTCTGGCAGCCATTGTTGGTTTTGAAAGCG
    GTCCGCGTATTGGTAAAGCAATTTATGGTAGC
    GATCTGCTGAATAATGGTTGGCATTGTGGTGC
    AGTTTATGGTGCACCGGCAGGCGCACTGGCCA
    CCGGTAAACTGCTGGGTCTGACACCGGATAGC
    ATGGAAGATGCACTGGGTATTGCCTGTACCCA
    GGCATGTGGTCTGATGAGCGCACAGTATGGTG
    GTATGGTTAAACGTGTTCAGCATGGTTTTGCA
    GCCCGTAATGGTCTGCTGGGTGGCCTGCTGGC
    ACATGGTGGTTATGAAGCAATGAAAGGTGTGC
    TGGAACGTAGCTATGGTGGTTTTCTGAAAATG
    TTTACCAAAGGCAATGGTCGTGAACCTCCGTA
    TAAAGAAGAAGAAGTIGTTGCAGGTCTGGGTA
    GCTTTTGGCATACCTTTACCATTCGCATTAAA
    CTGTATGCATGTTGTGGTCTGGTTCATGGTCC
    GGTGGAAGCAATTGAAAATCTGCAGGGTCGTT
    ATCCGGAACTGCTGAATCGTGCAAATCTGAGC
    AATATTCGTCATGTTCATGTTCAGCTGAGCAC
    CGCAAGCAATAGCCATTGCGGTTGGATTCCGG
    AAGAACGTCCGATTAGCAGCATTGCAGGTCAG
    ATGAGCGTTGCATATATTCTGGCCGTTCAGCT
    GGTTGATCAGCAGTGTCTGCTGAGCCAGTTTA
    GCGAATTTGATGATAACCTGGAACGTCCGGAA
    GTTTGGGATCTGGCACGTAAAGTTACCAGCAG
    CCAGAGCGAAGAATTTGATCAGGATGGTAATT
    GTCTGAGCGCAGGTCGTGTTCGTATTGAATTT
    AATGATGGTTCCAGCATTACCGAAAGCGTTGA
    AAAACCGCTGGGTGTTAAAGAACCGATGCCGA
    ATGAACGTATCCTGCATAAATATCGTACCCTG
    GCAGGTAGCGTTACCGATGAAAGCCGTGTGAA
    AGAAATTGAAGATCTGGTTCTGGGTCTGGATC
    GTCTGACCGATATTAGTCCGCTGCTGGAACTG
    CTGAACTGTCCGGTTAAAAGTCCGCTGGTTTA
    A
    3 AcnB W482R GTGCTAGAAGAATACCGTAAGCACGTAGCTGA
    GCGTGCCGCTGAGGGGATTGCGCCCAAACCCC
    TGGATGCAAACCAAATGGCCGCACTTGTAGAG
    CTGCTGAAAAACCCGCCCGCGGGCGAAGAAGA
    ATTCCTGTTAGATCTGTTAACCAACCGTGTTC
    CCCCTGGCGTCGATGAAGCCGCCTATGTCAAA
    GCAGGCTTCCTGGCTGCTATCGCGAAAGGCGA
    AGCCAAATCCCCTCTGCTGACTCCGGAAAAAG
    CCATCGAACTGCTGGGCACCATGCAGGGTGGT
    TACAACATTCATCCGCTGATCGACGCGCTGGA
    TGATGCCAAACTGGCACCGATCGCTGCCAAAG
    CACTTTCTCACACACTGCTGATGTTCGATAAC
    TTCTATGACGTAGAAGAGAAAGCGAAAGCAGG
    CAACGAATATGCGAAGCAGGTAATGCAGTCCT
    GGGCGGATGCCGAATGGTTCCTGAATCGCCCG
    GTGCTGGCTGAAAAACTGACCGTTACCGTCTT
    CAAAGTCACTGGCGAAACCAACACCGATGACC
    TTTCTCCGGCACCGGATGCGTGGTCGCGCCCG
    GATATCCCACTGCACGCGCTGGCGATGCTGAA
    AAACGCCCGTGAAGGTATTGAGCCAGACCAGC
    CAGGTGTTGTTGGCCCGATCAAACAGATTGAA
    GCTCTGCAACAGAAAGGTTTCCCGCTGGCGTA
    CGTCGGTGACGTTGTGGGTACGGGTTCATCGC
    GTAAATCCGCCACGAACTCCGTTCTGTGGTTT
    ATGGGCGATGATATTCCACATGTGCCGAACAA
    ACGCGGCGGTGGTTTGTGCCTCGGCGGTAAAA
    TTGCACCCATCTTCTTTAACACGATGGAAGAC
    GCGGGTGCACTGCCAATCGAAGTCGACGTCTC
    TAACCTGAACATGGGCGACGTGATTGACGTTT
    ACCCGTACAAAGGTGAAGTGCGTAACCACGAA
    ACAGGCGAACTGCTGGCGACCTTCGAACTGAA
    AACCGACGTGCTGATTGATGAAGTGCGTGCTG
    GCGGCCGTATCCCGCTGATTATCGGGCGTGGC
    CTGACCACCAAAGCGCGTGAAGCACTTGGTCT
    GCCGCACAGTGATGTGTTCCGTCAGGCGAAAG
    ATGTCGCTGAGAGCGATCGCGGCTTCTCGCTG
    GCGCAGAAAATGGTAGGCCGTGCCTGTGGCGT
    GAAAGGCATTCGTCCGGGCGCGTACTGCGAAC
    CGAAAATGACTTCTGTAGGCTCTCAGGACACC
    ACCGGCCCGATGACCCGTGATGAACTGAAAGA
    CCTGGCGTGCCTGGGCTTCTCGGCTGACCTGG
    TGATGCAGTCTTTCTGCCACACCGCGGCGTAT
    CCGAAGCCAGTTGACGTGAACACGCACCACAC
    GCTGCCGGACTTCATTATGAACCGTGGCGGTG
    TGTCGCTGCGTCCGGGTGACGGCGTCATTCAC
    TCCCGGCTGAACCGTATGCTGCTGCCGGATAC
    CGTCGGTACCGGTGGTGATTCCCATACCCGTT
    TCCCGATCGGTATCTCTTTCCCGGCGGGTTCT
    GGTCTGGTGGCATTTGCAGCCGCAACAGGCGT
    AATGCCGCTGGATATGCCGGAATCCGTTCTGG
    TGCGCTTCAAAGGCAAAATGCAGCCGGGCATC
    ACCCTGCGCGATCTGGTACATGCCATTCCGCT
    GTATGCGATCAAACAAGGTCTGCTGACCGTTG
    AGAAGAAAGGCAAGAAAAACATCTTCTCTGGC
    CGCATCCTGGAAATTGAAGGTCTGCCGGATCT
    GAAAGTTGAGCAGGCATTTGAACTGACCGATG
    CGTCCGCCGAGCGTTCTGCCGCTGGTTGTACC
    ATCAAGCTGAACAAAGAACCGATCATCGAATA
    CCTGAACTCTAACATCGTCCTGCTGAAGTGGA
    TGATCGCGGAAGGTTACGGCGATCGTCGCACC
    CTGGAACGTCGTATTCAGGGCATGGAAAAATG
    GCTGGCGAATCCTGAGCTGCTGGAAGCCGATG
    CAGATGCTGAATACGCGGCAGTGATCGACATC
    GATCTGGCGGATATTAAAGAGCCAATCCTCTG
    TGCACCGAACGACCCGGATGACGCGCGTCCGC
    TGTCTGCGGTACAGGGTGAGAAGATCGACGAA
    GTATTTATCGGTTCCTGCATGACCAACATCGG
    TCACTTCCGTGCTGCGGGTAAACTGCTGGATG
    CGCACAAAGGCCAACTGCCGACCCGCCTGTGG
    GTGGCACCGCCAACCCGTATGGACGCCGCGCA
    GTTGACTGAAGAAGGCTACTACAGCGTCTTCG
    GTAAGAGTGGTGCGCGTATCGAGATCCCGGGC
    TGTTCTCTGTGTATGGGTAACCAGGCGCGTGT
    AGCAGACGGTGCGACGGTGGTTTCCACCTCTA
    CCCGTAACTTCCCGAACCGTCTGGGTACTGGC
    GCGAATGTCTTCCTGGCTTCTGCGGAACTGGC
    GGCTGTTGCGGCGCTGATTGGCAAACTGCCGA
    CGCCGGAAGAGTACCAGACCTACGTGGCGCAA
    GTAGATAAAACTGCCGTTGATACTTATCGTTA
    TCTGAACTTCAACCAGCTTTCTCAGTACACCG
    AAAAAGCCGATGGGGTGATTTTCCAGACTGCG
    GTGTAA
    4 ic1R ATGGTCGCACCCATTCCCGCGAAACGCGGCAG
    AAAACCCGCCGTTGCCACCGCACCAGCGACTG
    GACAGGTTCAGTCTTTAACGCGTGGCCTGAAA
    TTACTGGAGTGGATTGCCGAATCCAATGGCAG
    TGTGGCACTCACAGAGCTGGCGCAACAAGCCG
    GGTTACCCAATTCCACGACCCACCGCCTGCTA
    ACCACGATGCAACAGCAGGGTTTCGTGCGTCA
    GGTCGGCGAACTGGGACATTGGGCAATCGGCG
    CACATGCCTTTATGGTCGGCAGCAGCTTTCTC
    CAGAGCCGTAATTTGTTAGCGATTGTTCACCC
    TATCCTGCGCAATTTAATGGAAGAGTCTGGCG
    AAACGGTCAATATGGCGGTGCTTGATCAAAGC
    GATCACGAAGCGATTATTATCGACCAGGTACA
    GTGTACGCATCTGATGCGAATGTCCGCGCCTA
    TCGGCGGTAAATTGCCGATGCACGCTTCCGGT
    GCGGGTAAAGCCTTTTTAGCCCAACTGAGCGA
    AGAACAGGTGACGAAGCTGCTGCACCGCAAAG
    GGTTACATGCCTATACCCACGCAACGCTGGTG
    TCTCCTGTGCATTTAAAAGAAGATCTCGCCCA
    AACGCGCAAACGGGGTTATTCATTTGACGATG
    AGGAACATGCACTGGGGCTACGTTGCCTTGCA
    GCGTGTATTTTCGATGAGCACCGCGAACCGTT
    TGCCGCAATTTCAATTTCCGGACCGATTTCAC
    GTATTACCGATGACCGCGTGACCGAGTTTGGC
    GCGATGGTGATTAAAGCGGCGAAGGAAGTGAC
    GCTGGCGTACGGTGGAATGCGCTGA
    5 Tac promoter ttgacaattaatcatcggctcgtataatg
    6 J23108 promoter ctgacagctagctcagtcctaggtataatgct
    agc
    7 PrpDV ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGgttGGCCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCAT
    CATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    8 PrpDT ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGTGGactCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCAT
    CATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    9 PrpDL ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGTGGGGCCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCct
    aATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    10 PrpDTL ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGtggactCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCct
    aATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    11 PrpDVT ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGgttactCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCat
    CATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    12 PrpDVL ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGgttggcCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCct
    aATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    13 J23115 promoter tttatagctagctcagcccttggtacaatgct
    agc
    14 J23114 promoter tttatggctagctcagtcctaggtacaatgct
    agc
    15 J23110 promoter tttacggctagctcagtcctaggtacaatgct
    agc
    16 J23100 promoter TTGACGGCTAGCTCAGTCCTAGGTACAGTGCT
    AGC
    17 PrpD wild type ATGTCAGCTCAAATCAACAACATCCGCCCGGA
    ATTTGATCGTGAAATCGTTGATATCGTCGATT
    ACGTCATGAACTACGAAATCAGCTCTAAAGTG
    GCCTACGACACCGCACATTACTGCCTGCTCGA
    CACGCTCGGCTGCGGTCTGGAAGCTCTCGAAT
    ACCCGGCCTGTAAAAAACTGCTGGGGCCAATT
    GTTCCCGGCACCGTCGTACCCAACGGCGTGCG
    CGTCCCCGGAACTCAGTTCCAGCTCGACCCCG
    TCCAGGCGGCATTTAACATCGGCGCGATGATC
    CGCTGGCTCGATTTCAACGATACCTGGCTGGC
    GGCGGAGTGGGGCCATCCTTCCGACAACCTCG
    GCGGCATTCTGGCAACGGCGGACTGGCTTTCG
    CGCAACGCGGTCGCCAGCGGCAAAGCGCCGTT
    GACCATGAAACAGGTGCTGACCGCAATGATCA
    AAGCCCATGAAATTCAGGGCTGCATCGCGCTG
    GAAAACTCCTTTAACCGCGTCGGCCTCGACCA
    CGTTCTGTTAGTGAAAGTGGCTTCCACCGCCG
    TGGTCGCCGAAATGCTCGGCCTGACCCGCGAG
    GAAATTCTCAACGCCGTTTCGCTGGCGTGGGT
    GGACGGTCAGTCGCTGCGCACCTATCGCCATG
    CGCCGAACACCGGCACGCGTAAATCCTGGGCG
    GCGGGCGATGCCACTTCCCGCGCGGTACGTCT
    GGCACTGATGGCGAAAACGGGCGAAATGGGTT
    ACCCGTCAGCCCTAACCGCGCCGGTATGGGGC
    TTCTACGACGTCTCCTTTAAAGGTGAATCGTT
    CCGCTTCCAGCGTCCGTACGGTTCTTACGTCA
    TGGAGAATGTGCTGTTCAAAATCTCCTTCCCG
    GCGGAGTTCCACTCCCAGACGGCAGTTGAAGC
    GGCGATGACGCTCTATGAACAGATGCAGGCAG
    CAGGCAAGACGGCGGCGGATATCGAAAAAGTG
    ACCATTCGCACCCACGAAGCCTGTATTCGCAT
    CATCGACAAAAAGGGGCCGCTCAATAACCCGG
    CTGACCGCGACCACTGCATTCAGTACATGGTG
    GCGATCCCGCTGTTATTCGGGCGCTTAACGGC
    GGCAGATTACGAGGACAACGTTGCGCAAGATA
    AACGCATCGACGCCCTGCGCGAGAAGATCAAT
    TGCTTTGAAGATCCGGCATTTACCGCTGACTA
    CCACGACCCGGAAAAACGCGCCATCGCCAATG
    CCATTACCCTTGAGTTCACCGACGGCACACGA
    TTTGAAGAAGTGGTGGTGGAGTACCCCATTGG
    TCATGCTCGCCGCCGTCAGGATGGTATTCCGA
    AACTGGTCGATAAATTCAAAATCAATCTCGCG
    CGCCAGTTCCCGACTCGCCAACAGCAGCGCAT
    TCTGGAGGTTTCTCTCGACAGAACTCGCCTGG
    AACAGATGCCGGTCAATGAGTATCTCGACCTG
    TACGTCATTTAA
    18 Synthetic 5′ UTR AAAAAAAACAAAAGGAGCATCACCC
    (Cad)
    19 Synthetic 5′ UTR CAACAAAAAAAAAGGAGCATCCTAC
    (PrpD and variant)
  • Collectively, the present inventors have improved the novel cis-aconitate synthesis enzyme and found that the new carbon flow to itaconate of the recombinant microorganisms introduced with the novel cis-aconitate synthesis enzyme was separated from the existing TCA cycle based on the activity of the enzyme, and finally the productivity of itaconate was increased. Therefore, the recombinant microorganism of the present disclosure can be variously used in the field of itaconate production.
  • As described above, specific parts of the present disclosure have been described in detail, and it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present disclosure is not limited thereto. Therefore, the substantial scope of the present disclosure will be defined by the appended claims and their equivalents.

Claims (14)

What is claimed is:
1. An expression cassette for introducing an itaconate production pathway comprising a 2-methylcitrate dehydratase (PrpD) gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17.
2. The expression cassette for introducing the itaconate production pathway of claim 1, further comprising:
a cis-aconitate decarboxylase (Cad) gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
3. The expression cassette for introducing the itaconate production pathway of claim 2, further comprising:
a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
4. The expression cassette for introducing the itaconate production pathway of claim 1, further comprising:
a synthetic 5′ untranslated region (5′ UTR) represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19.
5. The expression cassette for introducing the itaconate production pathway of claim 1, further comprising:
at least one promoter selected from the group consisting of SEQ ID NOs: 6, and 13 to 16.
6. A recombinant vector for producing itaconate comprising the expression cassette according to claim 1.
7. A recombinant microorganism for producing itaconate into which a PrpD gene represented by at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 7 to 12, and 17 is introduced.
8. The recombinant microorganism for producing itaconate of claim 7, further comprising:
a Cad gene represented by a nucleotide sequence set forth in SEQ ID NO: 2.
9. The recombinant microorganism for producing itaconate of claim 8, further comprising:
a Tac promoter represented by a nucleotide sequence set forth in SEQ ID NO: 5.
10. The recombinant microorganism for producing itaconate of claim 7, further comprising:
an aconitase B (AcnB) gene represented by a nucleotide sequence set forth in SEQ ID NO: 3.
11. The recombinant microorganism for producing itaconate of claim 7, wherein an iclR gene represented by a nucleotide sequence set forth in SEQ ID NO: 4 is deleted.
12. The recombinant microorganism for producing itaconate of claim 7, further comprising:
at least one promoter selected from the group consisting of SEQ ID NOs: 6, and 13 to 16.
13. The recombinant microorganism for producing itaconate of claim 7, further comprising:
a synthetic 5′ UTR represented by a nucleotide sequence set forth in SEQ ID NO: 18 or 19.
14. A method for producing itaconate comprising culturing the recombinant microorganism for producing itaconate of claim 7.
US18/192,868 2022-04-05 2023-03-30 Novel cis-aconitate synthesis enzyme and uses thereof Pending US20230313239A1 (en)

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KR20220042345 2022-04-05
KR10-2022-0042345 2022-04-05
KR10-2022-0189584 2022-12-29
KR1020220189584A KR20230143916A (en) 2022-04-05 2022-12-29 Novel cis-aconitate synthesis enzyme and uses thereof

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